This invention relates to a method of cancer screening and, more particularly, to a method primarily utilizing non-invasive cell collection techniques, and a combination of fluorescence detection techniques and radio tracing techniques for positive identification of malignant cells.
There are a number of prior art methods and apparatuses which are used in the detection and treatment of cancer. Fluorescent markers have been used to help identify cancerous tissue within a patient. Radio tracers or markers have also been used in the detection and treatment of cancer. There are also a number of prior art methods and apparatuses which relate to flow cytometry and the act of segregating and counting malignant cells within a tissue sample.
One example of a prior art reference which discloses the use of fluorescence detection for cancer screening is U.S. Pat. No. 5,270,171 to Cercek, et al. This reference teaches a method to identify, separate and purify the factor or factors that provoke a response by SCM (structuredness of the cytoplasmic matrix) responding lymphocytes. The use of such purified factor or factors enhances a SCM cancer screening test. The SCM is a peptide of at least nine amino acid residues. The residues produce at least a 10% decrease in the intracellular fluorescence polarization value of SCM responding lymphocytes from donors afflicted with cancer. Antibodies specific for the SCM factor are useful in immunoassays that can detect the factor, including detection of cancer cells grown in vitro. The SCM factor is useful for screening of blood samples and other body fluids or cell aspirates for the presence of malignancy in the donor. A method is also disclosed for testing lymphocytes obtained from the donor for presence or absence of a malignancy. A further method is also disclosed for screening a blood sample for the presence of a malignancy in a body of a donor.
U.S. Pat. No. 5,391,547 discloses a method of using porphyrins to detect lung cancer, and more particularly, to the use of tetra-aryl porphyrins. The porphyrins are used as a fluorescent tracer for cancers of the lung. The porphyrins may be complexed with Copper 64 (64Cu) or Copper 67 (67Cu). Thus, the complex can be used as radio tracers as well. The 67Cu provides a source of beta radiation for selective destruction of lung malignancies as well as gamma radiation useful for image analysis, as by single photon emission compute tomography. The 64Cu may be used for radio tracing wherein a positron emission tomography technique can be used to locate the malignant tissue.
U.S. Pat. No. 5,562,114 to Chu, et al. discloses a diagnostic immunoassay method using monoclonal antibodies. These monoclonal antibodies are capable of identifying an antigen associated with carcinomas of ductal lineage and can be used both diagnostically and therapeutically. More specifically, the monoclonal antibodies of this reference are capable of targeting the breast carcinoma cells in vivo. The monoclonal antibodies are purified and are labeled with radioactive compounds, for example, radioactive iodine, and then are administered to a patient intravenously. After a localization of the antibodies at the tumor site, they can be detected by emission, tomographical and radio nuclear scanning techniques thereby pinpointing the location of the cancer.
U.S. Pat. No. 5,087,636 to Jamieson, et al. discloses a method to identify and destroy malignant cells in mononuclear cell populations. This method includes the steps of contacting a composition of bone marrow cells or other cells with a green porphyrin of a specific compound, irradiating the cell composition with light at a wave length effective to excite fluorescence of the green porphyrin, and then detecting the presence or absence of fluorescence indicating malignancy. This reference also discloses the steps by which the bone marrow cells are removed, separated, washed and diluted to an appropriate concentration for treatment, incubated, centrifuged, and exposed to the irradiating light.
U.S. Pat. Nos. 5,308,608 and 5,149,708 to Dolphin, et al. disclose specific types of porphyrin compounds which may be used for detection, photosensitization, or the destruction of a targeted biological material when the targeted tissue is contacted with the specified porphyrin, and irradiated with light that excites the compound.
U.S. Pat. No. 5,211,938 to Kennedy, et al. discloses a method of detection of malignant and non-malignant lesions by photo chemotherapy of protoporphyrin IX precursors. 5-amino levulinic acid (5-ALA) is administered to the patient in an amount sufficient to induce synthesis of protoporphyrin IX in the lesions, followed by exposure of the treated lesion to a photo activating light in the range of 350-640 nanometers. Naturally occurring protoporphyrin IX is activatable by light which is in the incident red light range (600-700 nanometers) which more easily passes through human tissue as compared to light of other wave lengths which must be used with other types of porphyrins. In short, the use of 5-ALA makes cell fluorescence easier to observe, and also greatly reduces the danger of accidental phototoxic skin reactions in the days following treatment since protoporphyrin IX precursors have a much shorter half life in normal tissues than other popularly used porphyrins.
Another set of references exists which relate to flow cytometry utilizing fluorescence producing compounds. One such prior art reference includes U.S. Pat. No. 5,605,805 to Verwer, et al., which discloses a method for determining the lineage of acute leukemia cells in the sample by fluorocytometry. Other examples of fluorocytometry utilizing fluorescence include U.S. Pat. No. 5,418,169 to Crissman, et al., U.S. Pat. No. 5,556,764 to Sizto, et al., and U.S. Pat. No. 5,627,040 to Bierre.
Present methods relating to cancer screening using fluorescence detection systems require the use of interventional devices such as endoscopes which have the special capability of delivering specified light frequencies to a targeted area within a patient. These endoscopes illuminate the targeted part of the body in which cancer is suspected. The light delivered at a specified frequency illuminates an area which has previously been subjected to some type of fluorescent marker, such as a porphyrin which causes malignant cells to illuminate or fluoresce under observation of light at a specified frequency. In all cases, introduction of an endoscope into the body requires some type of sedation or general or local anesthesia. Once a tumor has been located by use of the interventional device, depending upon the type of tumor, photo chemotherapy or other treatment means can be used. However, prior to actual treatment, there must be a confirmed test of cancer. Accordingly, the tumor still needs to be sampled by an appropriate biopsy method. Generally, biopsy methods also require some type of sedation or anesthesia. Thus, traditional methods of confirming a malignancy may require at least two interventional surgical procedures.
While each of the foregoing references may be adequate for their intended purposes, many of these inventions require surgical techniques to remove the cell samples which can be traumatic to the patient. Furthermore, many of the references require complex equipment, and special medical expertise in order to conduct the procedures and to make the diagnoses. Therefore, there is a need for a reliable cancer screening technique or method which can test for cancer in a wide variety of cells and which may be accomplished by non-invasive or minimally invasive cell collection techniques which limit patient trauma, are inexpensive to conduct, and can be confirmed positively by a pathologist, oncologist or other physician without additional testing or screening. There is also a need for a reliable cancer screening technique or method which can utilize two different types of screening technologies within a single cancer screening procedure, therefore improving the ability to make a diagnosis of cancer, and also to provide a follow-on treatment of the cancer, either through photo-dynamic therapy and/or through radiation-treatment. The invention described below provides each of these advantages, among others, which will be apparent to those skilled in the art.
The present invention relates to a method of cancer screening utilizing non-invasive or minimally invasive cell collection techniques, fluorescence detection techniques, and radio detection techniques. The invention herein also contemplates treatment of a cancer by various photo therapy treatments and/or radiation treatments.
The term xe2x80x9cnon-invasivexe2x80x9d as used herein and as applied to a specific cell collection technique shall mean cell collection which does not involve the forced removal of tissue as by the act of cutting or otherwise tearing away cell tissue which would normally remain attached to the body. As discussed further below, a cell collection technique using a cytological brush would be considered non-invasive because, although contact is made with a targeted area of tissue to be removed, the cytological brush simply removes a top layer(s) of cells which would normally exfoliate or desquamate from the body. Thus, a cytological brush used according to the cell collection techniques of this invention does not involve the scraping of tissue to a degree that it cuts or tears tissue away from the body. The term xe2x80x9cminimally invasivexe2x80x9d as used herein and as applied to other cell collection techniques disclosed herein shall mean the removal of cells from the body which requires some interventional means for accessing the targeted group of cells, but does not require the actual tearing or cutting away of such targeted tissue. As discussed further below, minimally invasive cell collection techniques include the use of a fine gauge needle or catheter which must penetrate the body to gain access to interior targeted tissue. This minimally invasive cell collection technique is used specifically with the collection of cells from the central nervous system, peritoneal cavity, and thoracic cavity. Cell collection from these areas in the body is not achieved by cutting or tearing away the tissue, but is achieved by non-invasive means once the minimally invasive access procedure has taken place.
Thus, according to one aspect of the present invention, the exfoliation or dislodgement of cells from the human body is achieved through non-invasive means. For dislodgement of pulmonary system cells, techniques are disclosed which include fist percussion while a patient is placed in a postural drainage position. For exfoliation of gastrointestinal cells, the techniques include lavage cytology by oral administration of a first balanced electrolyte solution to cleanse the bowel which is followed by oral administration of an additional electrolyte solution to produce a clear anal effluent for cytologic evaluation. Cells in the oral cavity may be collected by a cytological brush. For prostate gland cell dislodgement, a physician may xe2x80x9cmilkxe2x80x9d the prostate to express contained fluids which are carried through the ductal system to the urethra via the seminal vesicles and the ejaculatory ducts. For urinary tract cells, exfoliation may be achieved by rapid oral fluid intake and the use of a diuretic such as Lasix(trademark). For collection of cervical and uterine cell samples, a cytological brush may also be used. For breast cell collection, the ductal system of the breast may be opened by the use of a product such as Seruminex(trademark) which dissolves xe2x80x9cplugsxe2x80x9d in the ducts of the nipple, and gravity is allowed to cause fluids to drain out. The discussion below more fully details these special non-invasive cell collection techniques. Collection of other cell types is also discussed below.
Once the targeted cells have been removed from the body, they are immediately placed in a temperature controlled (37xc2x0 C.) cell culture solution or media to keep them alive a desired period of time. For most cells, a cell transport media is used which is identical to commercially available cell culture media. A water bath is typically used to maintain the culture at the desired temperature.
A photosensitive compound is then introduced to the cell culture. These compounds when administered in appropriate amounts selectively enter into pre-malignant and malignant cells, and provide a xe2x80x9cfluorescent markerxe2x80x9d in the cells, primarily in the mitochondria surrounding the nucleus. The compounds which may be used in this method to induce fluorescence include 5-ALA, protoporphyrin IX, tetrakis carboxy-phenyl porphine (TCPP), hematoporphyrine derivative, photofrin, and photofrin II and other known in the art to cause fluorescence in pre-malignant or malignant cells. For TCPP, this compound enters live cells via a special transport mechanism found in the outer cellular wall. TCPP will not enter dead cells, thus making it important that a live cell culture be maintained. Once inside the cell, TCPP appears to migrate to the perinuclear areas and become involved with the mitochondria. In short, the above compounds will cause pre-malignant or malignant cells to fluoresce when exposed to frequencies of light which match the excitation frequency of the particular compound used; however healthy cells will generally not fluoresce.
Once the photosensitive compounds are introduced to the cell culture, they are allowed to interact with the cell tissue a specified amount of time in a controlled environment. After this incubation period, cells may be examined by use of a fluorescence microscope to see if any cells fluoresce. Fluorescence in the cell indicates a high degree of suspicion for malignancy. The cell culture can first be centrifuged to help separate the cells from the cell culture fluids. The cells are resuspended in saline, and a small aliquot is placed on a slide. If no cells are found to fluoresce after initial observation under the fluorescence microscope, the cells are disaggregated and processed through a flow cytometer utilizing fluorescence detection. This is done to ensure that no fluorescent cells are overlooked. Manual examination of cell suspensions is not particularly accurate, since millions of cells need to be examined. Flow cytometers can find a single fluorescent cell in a field of millions of cells with virtual 100% accuracy. The fluorescence microscope and the flow cytometer provide light to match the excitation frequency of the particular compound used. For example, the excitation frequency for TCPP is approximately 380-450 nanometers. If fluorescent malignant cells are found by the fluorescence microscope, they may also be counted and disaggregated for further study. After incubation, no further care of the cell specimen is required.
Alternatively, the above-described photosensitive compounds may be administered directly to the patient prior to cell collection. 5-ALA can be administered orally, topically, or parenterally (by injection); however, the other compounds have to be administered topically or by injection (parenterally). The waiting period prior to cell collection is then two to four hours, depending upon the compound introduced. After sufficient time has been provided for interaction between the compound and the targeted cells, the cells may then be exfoliated or dislodged from the patient through non-invasive or minimally invasive techniques.
In addition to providing the above described photo sensitive compounds alone, the photo sensitive compounds may be complexed with a radioisotope, such as 64Cu or 67Cu. Accordingly, these complexes then provide the ability for not only conducting fluorescence detection and photo-therapy, but also provides the ability for observation of malignancies through positron emission tomography (for 64Cu) or single photon emission computed tomography (for 67Cu). Additionally, for 67Cu, it provides a source of beta radiation for selective destruction of malignancies. As well understood by those skilled in the art, positron emission tomography (PET) is a scanning technique which enables precise mapping of tissue which uptakes the radioactive marker. Single photon emission computed tomography (SPECT) is closely related to PET scanning and also provides an exact means to locate targeted tissue which uptakes an introduced radio tracer.
For those cells which are not dislodged through exfoliation and are therefore left within the body, those cells are doubly tagged not only for fluorescence, but for radioactivity as well. It may be, therefore, desirable under some circumstances to not only remove cells from the body for exuluo analysis, but also to investigate believed malignancies through fluorescence guided endoscopy, or to view the suspect cells through the appropriate tomography technique. One advantage of attaching a radioisotope to the photosensitive compounds is that through the use of recently available tomography devices one can, in effect, conduct a complete body scan of all potentially malignant areas within the body without having to conduct separate endoscopic procedures for each potentially malignant site found. The images taken from the scanning technique can then be provided to the particular specialist for follow-on treatment, which may include surgical intervention or radiation treatment.
By the method of this invention, a quick and reliable means is provided for cancer screening. Because non-invasive or minimally invasive techniques are used for cell collection, patient trauma is reduced along with the cost of the procedure. Because the method of this invention provides the option of introducing the compounds ex-vivo, the concern for any possible allergic reaction or phototoxic reaction by a patient""s exposure to the sun is eliminated. Furthermore, because no tissue biopsies are taken, the method of this invention eliminates the inherent hazard in administering local and/or general anesthesia. Cell marking by use of the above-identified compounds is extremely reliable in terms of differentiating healthy cells from pre-malignant or malignant cells. The segregation, counting and analysis of the fluorescent cells may be achieved with commercially available flow cytometers and supporting equipment. The results of the cancer screening procedure may be forwarded to a pathologist who may wish to conduct additional tests to further determine the exact nature of the malignancy. The fluorescing cells may be photographed to provide documentation of malignancy.
The method of the invention also provides the option of utilizing radioactively tagged photo-sensitive compounds, wherein fluorescence guided endoscopy can be conducted for viewing and treatment of a malignancy, and additionally, the radioactive tag allows various tomography techniques to be used for further visualization of potentially malignant tissue as well as providing a means for radioactive treatment. These and other advantages are discussed more fully below in the detailed description taken in conjunction with the corresponding figures.